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Underexpression, overexpression, and point mutations in peripheral myelin protein 22 (PMP22) cause most cases of Charcot-Marie-Tooth disease (CMTD). While its exact functions remain unclear, PMP22 is clearly essential for formation and maintenance of healthy myelin in the peripheral nervous system. This review explores emerging evidence for roles of PMP22 in cholesterol homeostasis. First, we highlight dysregulation of lipid metabolism in PMP22-based forms of CMTD and recently-discovered interactions between PMP22 and cholesterol biosynthesis machinery. We then examine data that demonstrates PMP22 and cholesterol co-traffic in cells and co-localize in lipid rafts, including how disease-causing PMP22 mutations result in aberrations in cholesterol localization. Finally, we examine roles for interactions between PMP22 and ABCA1 in cholesterol efflux. Together, this emerging body of evidence suggests that PMP22 plays a role in facilitating enhanced cholesterol synthesis and trafficking necessary for production and maintenance of healthy myelin., (© 2024 The Author(s).)

Snakebite is a major global health concern, for which antivenom remains the only approved treatment to neutralise the harmful effects of the toxins. However, some medically important toxins are poorly immunogenic, resulting in reduced efficacy of the final product. Boosting the immunogenicity of these toxins in the commercial antivenom immunising mixtures could be an effective strategy to improve the final dose efficacy, and displaying snake antigens on Virus-like particles (VLPs) is one method for this. However, despite some applications in the field of snakebite, VLPs have yet to be explored in methods that could be practical at an antivenom manufacturing scale. Here we describe the utilisation of a "plug and play" VLP system to display immunogenic linear peptide epitopes from three finger toxins (3FTxs) and generate anti-toxin antibodies. Rabbits were immunised with VLPs displaying individual consensus linear epitopes and their antibody responses were characterised by immunoassay. Of the three experimental consensus sequences, two produced antibodies capable of recognising the consensus peptides, whilst only one of these could also recognise native whole toxins. Further characterisation of antibodies raised against this peptide demonstrated a sub-class specific response, and that these were able to elicit partially neutralising antibody responses, resulting in increased survival times in a murine snakebite envenoming model., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Simon J Draper reports a relationship with SpyBiotech that includes: board membership and equity or stocks. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)

Snake venoms are cocktails of biologically active molecules that have evolved to immobilize prey, but can also induce a severe pathology in humans that are bitten. While animal-derived polyclonal antivenoms are the primary treatment for snakebites, they often have limitations in efficacy and can cause severe adverse side effects. Building on recent efforts to develop improved antivenoms, notably through monoclonal antibodies, requires a comprehensive understanding of venom toxins. Among these toxins, snake venom metalloproteinases (SVMPs) play a pivotal role, particularly in viper envenomation, causing tissue damage, hemorrhage and coagulation disruption. One of the current challenges in the development of neutralizing monoclonal antibodies against SVMPs is the large size of the protein and the lack of existing knowledge of neutralizing epitopes. Here, we screened a synthetic human antibody library to isolate monoclonal antibodies against an SVMP from saw-scaled viper (genus Echis ) venom. Upon characterization, several antibodies were identified that effectively blocked SVMP-mediated prothrombin activation. Cryo-electron microscopy revealed the structural basis of antibody-mediated neutralization, pinpointing the non-catalytic cysteine-rich domain of SVMPs as a crucial target. These findings emphasize the importance of understanding the molecular mechanisms of SVMPs to counter their toxic effects, thus advancing the development of more effective antivenoms.

Snakebites affect about 1.8 million people annually. The current standard of care involves antibody-based antivenoms, which can be difficult to access and are generally not effective against local tissue injury, the primary cause of morbidity. Here, we used a pooled whole-genome CRISPR knockout screen to define human genes that, when targeted, modify cell responses to spitting cobra venoms. A large portion of modifying genes that conferred resistance to venom cytotoxicity was found to control proteoglycan biosynthesis, including EXT1 , B4GALT7 , EXT2 , EXTL3 , XYLT2 , NDST1 , and SLC35B2 , which we validated independently. This finding suggested heparinoids as possible inhibitors. Heparinoids prevented venom cytotoxicity through binding to three-finger cytotoxins, and the US Food and Drug Administration-approved heparinoid tinzaparin was found to reduce tissue damage in mice when given via a medically relevant route and dose. Overall, our systematic molecular dissection of cobra venom cytotoxicity provides insight into how we can better treat cobra snakebite envenoming.

Background: Despite the high incidence of blunt trauma in older adults, there is a lack of evidence-based guidance for computed tomography (CT) imaging in this population. We aimed to identify an algorithm to guide use of a Pan-Scan (Head/C-spine/Torso) or a Selective Scan (Head/C-spine ± Torso). We hypothesized that a patient's initial history and exam could be used to guide imaging., Methods: We prospectively studied blunt trauma patients aged 65+ at 18 Level I/II trauma centers. Patients presenting >24 h after injury or who died upon arrival were excluded. We collected history and physical elements and final injury diagnoses. Injury diagnoses were categorized into CT body regions of Head/C-spine or Torso (chest, abdomen/pelvis, and T/L spine). Using machine learning and regression modeling as well as a priori clinical algorithms based, we tested various decision rules against our dataset. Our priority was to identify a simple rule which could be applied at the bedside, maximizing sensitivity (Sens) and negative predictive value (NPV) to minimize missed injuries., Results: We enrolled 5,498 patients with 3,082 injuries. Nearly half (47.1%, n = 2,587) had an injury within the defined CT body regions. No rule to guide a Pan-Scan could be identified with suitable Sens/NPV for clinical use. A clinical algorithm to identify patients for Pan-Scan, using a combination of physical exam findings and specific high-risk criteria, was identified and had a Sens of 0.94 and NPV of 0.86 This rule would have identified injuries in all but 90 patients (1.6%) and would theoretically spare 11.9% (655) of blunt trauma patients a torso CT., Conclusions: Our findings advocate for Head/Cspine CT in all geriatric patients with the addition of torso CT in the setting of positive clinical findings and high-risk criteria. Prospective validation of this rule could lead to streamlined diagnostic care of this growing trauma population., Level of Evidence: Level 2, Diagnostic Tests or Criteria., Competing Interests: Conflict of Interest: All JTACS Disclosure forms have been supplied and are provided as supplemental digital content (http://links.lww.com/TA/D870)., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

Snakebite envenoming is a neglected tropical disease that causes substantial mortality and morbidity globally. The venom of African spitting cobras often causes permanent injury via tissue-destructive dermonecrosis at the bite site, which is ineffectively treated by current antivenoms. To address this therapeutic gap, we identified the etiological venom toxins in Naja nigricollis venom responsible for causing local dermonecrosis. While cytotoxic three-finger toxins were primarily responsible for causing spitting cobra cytotoxicity in cultured keratinocytes, their potentiation by phospholipases A 2 toxins was essential to cause dermonecrosis in vivo. This evidence of probable toxin synergism suggests that a single toxin-family inhibiting drug could prevent local envenoming. We show that local injection with the repurposed phospholipase A 2 -inhibiting drug varespladib significantly prevents local tissue damage caused by several spitting cobra venoms in murine models of envenoming. Our findings therefore provide a therapeutic strategy that may effectively prevent life-changing morbidity caused by snakebite in rural Africa., Competing Interests: Competing interests statement:The authors declare no competing interest.

Introduction: Antivenom is a lifesaving medicine for treating snakebite envenoming, yet there has been a crisis in antivenom supply for many decades. Despite this, substantial quantities of antivenom stocks expire before use. This study has investigated whether expired antivenoms retain preclinical quality and efficacy, with the rationale that they could be used in emergency situations when in-date antivenom is unavailable., Methods: Using WHO guidelines and industry test requirements, we examined the in vitro stability and murine in vivo efficacy of eight batches of the sub-Saharan African antivenom, South African Institute for Medical Research polyvalent, that had expired at various times over a period of 30 years., Results: We demonstrate modest declines in immunochemical stability, with antivenoms older than 25 years having high levels of turbidity. In vitro preclinical analysis demonstrated all expired antivenoms retained immunological recognition of venom antigens and the ability to inhibit key toxin families. All expired antivenoms retained comparable in vivo preclinical efficacy in preventing the lethal effects of envenoming in mice versus three regionally and medically important venoms., Conclusions: This study provides strong rationale for stakeholders, including manufacturers, regulators and health authorities, to explore the use of expired antivenom more broadly, to aid in alleviating critical shortages in antivenom supply in the short term and the extension of antivenom shelf life in the longer term., Competing Interests: Competing interests: GS, GD, AS, MV and GL are all employees of Instituto Clodomiro Picado, University of Costa Rica, a public manufacturer of antivenom products, including for sub-Saharan Africa., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY. Published by BMJ.)

Antimicrobial peptides (AMPs) are at the interface of interactions between hosts and microbes and are therefore expected to be rapidly evolving in a coevolutionary arms race with pathogens. In contrast, previous work demonstrated that insect AMPs tend to evolve more slowly than the genome average. Metchikowin (Mtk) is a Drosophila AMP that has a single amino acid residue that segregates as either proline (P) or arginine (R) in populations of four different species, some of which diverged more than 10 million years ago. These results suggest that there is a distinct functional importance to each allele. The most likely hypotheses are driven by two main questions: does each allele have a different efficacy against different specific pathogens (specificity hypothesis)? Or, is one allele a more potent antimicrobial, but with a host fitness cost (autoimmune hypothesis)? To assess their functional differences, we created D. melanogaster lines with the P allele, R allele, or Mtk null mutation using CRISPR/Cas9 genome editing and performed a series of life history and infection assays to assess them. In males, testing of systemic immune responses to a repertoire of bacteria and fungi demonstrated that the R allele performs as well or better than the P and null alleles with most infections. Females show some results that contrast with males, with Mtk alleles either not contributing to survival or with the P allele outperforming the R allele. In addition, measurements of life history traits demonstrate that the R allele is more costly in the absence of infection for both sexes. These results are consistent with both the specificity hypothesis (either allele can perform better against certain pathogens depending on context), and the autoimmune hypothesis (the R allele is generally the more potent antimicrobial in males, and carries a fitness cost). These results provide strong in vivo evidence that differential fitness with or without infection and sex-based functional differences in alleles may be adaptive mechanisms of maintaining immune gene polymorphisms in contrast with expectations of rapid evolution. Therefore, a complex interplay of forces including pathogen species and host sex may lead to balancing selection for immune genotypes. Strikingly, this selection may act on even a single amino acid polymorphism in an AMP., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Perlmutter et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Antivenom is currently the first-choice treatment for snakebite envenoming. However, only a low proportion of antivenom immunoglobulins are specific to venom toxins, resulting in poor dose efficacy and potency. We sought to investigate whether linear venom epitopes displayed on virus like particles can stimulate an antibody response capable of recognising venom toxins from diverse medically important species. Bioinformatically-designed epitopes, corresponding to predicted conserved regions of group I phospholipase A 2 and three finger toxins, were engineered for display on the surface of hepatitis B core antigen virus like particles and used to immunise female CD1 mice over a 14 weeks. Antibody responses to all venom epitope virus like particles were detectable by ELISA by the end of the immunisation period, although total antibody and epitope specific antibody titres were variable against the different epitope immunogens. Immunoblots using pooled sera demonstrated recognition of various venom components in a diverse panel of six elapid venoms, representing three continents and four genera. Insufficient antibody yields precluded a thorough assessment of the neutralising ability of the generated antibodies, however we were able to test polyclonal anti-PLA 2 IgG from three animals against the PLA 2 activity of Naja nigricollis venom, all of which showed no neutralising ability. This study demonstrates proof-of-principle that virus like particles engineered to display conserved toxin linear epitopes can elicit specific antibody responses in mice which are able to recognise a geographically broad range of elapid venoms., (© 2022. The Author(s).)

Snakebite is a neglected tropical disease that causes high rates of global mortality and morbidity. Although snakebite can cause a variety of pathologies in victims, haemotoxic effects are particularly common and are typically characterised by haemorrhage and/or venom-induced consumption coagulopathy. Despite polyclonal antibody-based antivenoms being the mainstay life-saving therapy for snakebite, they are associated with limited cross-snake species efficacy, as there is often extensive toxin variation between snake venoms, including those used as immunogens for antivenom production. This restricts the therapeutic utility of any antivenom to certain geographical regions. In this study, we explored the feasibility of using recombinantly expressed toxins as immunogens to stimulate focused, pathology-specific, antibodies in order to broadly counteract specific toxins associated with snakebite envenoming. Three snake venom serine proteases (SVSP) toxins, sourced from geographically diverse and medically important viper snake venoms, were successfully expressed in HEK293F mammalian cells and used for murine immunisation. Analyses of the resulting antibody responses revealed that ancrod and RVV-V stimulated the strongest immune responses, and that experimental antivenoms directed against these recombinant SVSP toxins, and a mixture of the three different immunogens, extensively recognised and exhibited immunological binding towards a variety of native snake venoms. While the experimental antivenoms showed some reduction in abnormal clotting parameters stimulated by the toxin immunogens and crude venom, specifically reducing the depletion of fibrinogen levels and prolongation of prothrombin times, fibrinogen degradation experiments revealed that they broadly protected against venom- and toxin-induced fibrinogenolytic functional activities. Overall, our findings further strengthen the case for the use of recombinant venom toxins as supplemental immunogens to stimulate focused and desirable antibody responses capable of neutralising venom-induced pathological effects, and therefore potentially circumventing some of the limitations associated with current snakebite therapies.

Background: Venoms are ecological innovations that have evolved numerous times, on each occasion accompanied by the co-evolution of specialised morphological and behavioural characters for venom production and delivery. The close evolutionary interdependence between these characters is exemplified by animals that control the composition of their secreted venom. This ability depends in part on the production of different toxins in different locations of the venom gland, which was recently documented in venomous snakes. Here, we test the hypothesis that the distinct spatial distributions of toxins in snake venom glands are an adaptation that enables the secretion of venoms with distinct ecological functions., Results: We show that the main defensive and predatory peptide toxins are produced in distinct regions of the venom glands of the black-necked spitting cobra (Naja nigricollis), but these distributions likely reflect developmental effects. Indeed, we detected no significant differences in venom collected via defensive 'spitting' or predatory 'biting' events from the same specimens representing multiple lineages of spitting cobra. We also found the same spatial distribution of toxins in a non-spitting cobra and show that heterogeneous toxin distribution is a feature shared with a viper with primarily predatory venom., Conclusions: Our findings suggest that heterogeneous distributions of toxins are not an adaptation to controlling venom composition in snakes. Instead, it likely reflects physiological constraints on toxin production by the venom glands, opening avenues for future research on the mechanisms of functional differentiation of populations of protein-secreting cells within adaptive contexts., (© 2022. The Author(s).)

This work reports substrate-selective inhibition of a protease with broad substrate specificity based on direct binding of a small-molecule inhibitor to the substrate. The target for these studies was γ-secretase protease, which cleaves dozens of different single-span membrane protein substrates, including both the C99 domain of the human amyloid precursor protein and the Notch receptor. Substrate-specific inhibition of C99 cleavage is desirable to reduce production of the amyloid-β polypeptide without inhibiting Notch cleavage, a major source of toxicity associated with broad specificity γ-secretase inhibitors. In order to identify a C99-selective inhibitors of the human γ-secretase, we conducted an NMR-based screen of FDA-approved drugs against C99 in model membranes. From this screen, we identified the small-molecule verteporfin with these properties. We observed that verteporfin formed a direct 1:1 complex with C99, with a K D of 15-47 μM (depending on the membrane mimetic used), and that it did not bind the transmembrane domain of the Notch-1 receptor. Biochemical assays showed that direct binding of verteporfin to C99 inhibits γ-secretase cleavage of C99 with IC 50 values in the range of 15-164 μM, while Notch-1 cleavage was inhibited only at higher concentrations, and likely via a mechanism that does not involve binding to Notch-1. This work documents a robust NMR-based approach to discovery of small-molecule binders to single-span membrane proteins and confirmed that it is possible to inhibit γ-secretase in a substrate-specific manner., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

The SARS-CoV-2 pandemic from 2019 onwards has significantly increased the usage of surgical style medical masks, both in healthcare and public settings. It is important to study the contamination of and viral transfer from such masks. However, accepted standard test methods such as ISO 18184 have prescribed inoculation methods which may not be fully representative of the type of viral insult experienced in the clinic or community. In addition to studying a conventional mask, the performance of a mask featuring an antimicrobial photosensitiser was also studied., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2022 Mark C. Wilkinson and Jennifer Carney.)

Bites from elapid snakes typically result in neurotoxic symptoms in snakebite victims. Neurotoxins are, therefore, often the focus of research relating to understanding the pathogenesis of elapid bites. However, recent evidence suggests that some elapid snake venoms contain anticoagulant toxins which may help neurotoxic components spread more rapidly. This study examines the effects of venom from the West African black-necked spitting cobra ( Naja nigricollis ) on blood coagulation and identifies potential coagulopathic toxins. An integrated RPLC-MS methodology, coupled with nanofractionation, was first used to separate venom components, followed by MS, proteomics and coagulopathic bioassays. Coagulation assays were performed on both crude and nanofractionated N. nigricollis venom toxins as well as PLA 2 s and 3FTx purified from the venom. Assays were then repeated with the addition of either the phospholipase A 2 inhibitor varespladib or the snake venom metalloproteinase inhibitor marimastat to assess whether either toxin inhibitor is capable of neutralizing coagulopathic venom activity. Subsequent proteomic analysis was performed on nanofractionated bioactive venom toxins using tryptic digestion followed by nanoLC-MS/MS measurements, which were then identified using Swiss-Prot and species-specific database searches. Varespladib, but not marimastat, was found to significantly reduce the anticoagulant activity of N. nigricollis venom and MS and proteomics analyses confirmed that the anticoagulant venom components mostly consisted of PLA 2 proteins. We, therefore, conclude that PLA 2 s are the most likely candidates responsible for anticoagulant effects stimulated by N. nigricollis venom.

Snakebite envenoming causes 138,000 deaths annually, and ~400,000 victims are left with permanent disabilities. Envenoming by saw-scaled vipers (Viperidae: Echis ) leads to systemic hemorrhage and coagulopathy and represents a major cause of snakebite mortality and morbidity in Africa and Asia. The only specific treatment for snakebite, antivenom, has poor specificity and low affordability and must be administered in clinical settings because of its intravenous delivery and high rates of adverse reactions. This requirement results in major treatment delays in resource-poor regions and substantially affects patient outcomes after envenoming. Here, we investigated the value of metal ion chelators as prehospital therapeutics for snakebite. Among the tested chelators, dimercaprol (British anti-Lewisite) and its derivative 2,3-dimercapto-1-propanesulfonic acid (DMPS) were found to potently antagonize the activity of Zn 2+ -dependent snake venom metalloproteinases in vitro. Moreover, DMPS prolonged or conferred complete survival in murine preclinical models of envenoming against a variety of saw-scaled viper venoms. DMPS also considerably extended survival in a "challenge and treat" model, where drug administration was delayed after venom injection and the oral administration of this chelator provided partial protection against envenoming. Last, the potential clinical scenario of early oral DMPS therapy combined with a delayed, intravenous dose of conventional antivenom provided prolonged protection against the lethal effects of envenoming in vivo. Our findings demonstrate that the safe and affordable repurposed metal chelator DMPS can effectively neutralize saw-scaled viper venoms in vitro and in vivo and highlight the promise of this drug as an early, prehospital, therapeutic intervention for hemotoxic snakebite envenoming., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Objective: The purpose of this study was to design and implement a realistic, durable, and low-cost training model for percutaneous renal access., Material and Methods: Ballistic gelatin mixed with radiographic contrast was poured into surgical gloves to create a radio-dense renal collecting system. The collecting system model was then embedded in a pure ballistic gelatin block resting upon a clear acrylic glass base. Finally, the model was covered by a visually opaque polyurethane foam cover with chalk sticks positioned to simulate ribs. Experienced attending urologists and interventional radiologists, urology residents, and medical students used the model to access the upper, middle, and lower renal calyces under fluoroscopic guidance. Outcomes included model durability, realism rated by participants on a visual analogue scale, and cost., Results: The ballistic gelatin model was durable and anatomically realistic. Each model sustained over 200 needle punctures with no significant compromise in structural integrity or any contrast leakage. Attending and resident physicians considered it to provide an accurate simulation of renal access and medical students and residents considered the model to be a practical training modality (residents 8.4/10 vs. medical students 9.4/10). The total cost for one model was $60., Conclusion: The ballistic gelatin collecting system provided a realistic, durable, and low-cost renal access training model. This could allow trainees to develop skills without compromising patient safety.

Immunoglobulin light chain amyloidosis is the most common form of systemic amyloidosis. However, very little is known about the underlying mechanisms that initiate and modulate the associated protein aggregation and deposition. Model systems have been established to investigate these disease-associated processes. One of these systems comprises two 114 amino acid light-chain variable domains of the kappa 4 IgG family, SMA and LEN. Despite high sequence identity (93%), SMA is amyloidogenic in vivo, but LEN adopts a stable dimer, displaying amyloidogenic properties only under destabilising conditions in vitro. We present here a refined and reproducible periplasmic expression and purification protocol for SMA and LEN that improves on existing methods and provides high yields of pure protein (10-50mg/L), particularly suitable for structural studies that demand highly concentrated and purified proteins. We confirm that recombinant SMA and LEN proteins have structure and dimerization capabilities consistent with the native proteins and employ fluorescence to probe internalization and cellular localization within cardiomyocytes. We propose periplasmic expression and simplified chromatographic steps outlined here as an optimized method for production of these and other variable light chain domains to investigate the underlying mechanisms of light chain amyloidosis. We show that SMA and LEN can be internalised within cardiomyocytes and were observed to localise to the perinuclear area, assessed by confocal microscopy as a possible mechanism for underlying cytotoxicity and pathogenesis associated with amyloidosis., Competing Interests: The authors have declared that no competing interests exist.

Type III protein secretion systems (T3SS) are encoded by several pathogenic or symbiotic bacteria. The central component of this nanomachine is the needle complex. Here we show in a Salmonella Typhimurium T3SS that assembly of the needle filament of this structure requires OrgC, a protein encoded within the T3SS gene cluster. Absence of OrgC results in significantly reduced number of needle substructures but does not affect needle length. We show that OrgC is secreted by the T3SS and that exogenous addition of OrgC can complement a ∆orgC mutation. We also show that OrgC interacts with the needle filament subunit PrgI and accelerates its polymerization into filaments in vitro. The structure of OrgC shows a novel fold with a shared topology with a domain from flagellar capping proteins. These findings identify a novel component of T3SS and provide new insight into the assembly of the type III secretion machine., Competing Interests: JK, SD, JS, CB, MW, RD, JG No competing interests declared, (© 2018, Kato et al.)

Snake venom metalloproteinases (SVMPs) are the key enzymes in Russell's viper (RV) venom which target all important components of haemostasis, such as clotting factors, platelets, endothelial cells and basement membrane. The structural diversity of SVMPs contributes to the broad spectrum of biological activities. The aim of the study was to investigate the SVMP transcript profile to gain better insights into the characteristic clinical manifestations of the Myanmar Russell's viper (MRV) bites that distinguish it from the RVs of other habitats. Next generation sequencing (RNA-Seq) of mRNA from MRV venom glands (2 males and 1 female) was performed on an Illumina HiSeq2000 platform and then de novo assembled using Trinity software. A total of 59 SVMP contigs were annotated through a Blastn search against the serpent nucleotide database from NCBI. Among them, disintegrins were the most abundant transcripts (75%) followed by the P-III class SVMPs (25%). The P-II SVMPs were scarce (0.002%), while no P-I SVMPs were detectable in the transcriptome. For detailed structural analysis, contigs were conceptually translated and compared with amino acid sequences from other RVs and other vipers using Clustal Omega. The RTS-disintegrin (jerdostatin homolog) was the most abundant among transcripts corresponding to 5 disintegrin isoforms. From 10 isoforms of SVMPs, RVV-X, and Vipera lebetina apoptosis-inducing protease (VLAIP) homolog, hereby termed Daboia siamensis AIP (DSAIP), were found to be highly expressed. Venom protein analysis using SDS-PAGE followed by mass spectrometry revealed that the disintegrin was scarce, while the latter two SVMPs were abundant. These two proteins can contribute to severe clinical manifestations caused by MRV envenomation., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Three aspartic proteases (SVAPs) have been isolated from venom of the saw-scaled viper, Echis ocellatus. In confirmation of prior transcriptomic predictions, all three forms match to sequences of either of the two SVAP transcripts (EOC00051 and EOC00123), have a molecular weight of 42 kDa and possess a single N-glycan. The SVAPs act in a renin-like manner, specifically cleaving human and porcine angiotensinogen into angiotensin-1 and possess no general protease activity. Their activity is completely inhibited by the aspartyl protease inhibitor Pepstatin A., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Purpose: Postoperative hemoglobin (Hb) determination remains an essential parameter for quantifying blood loss following total knee replacement (TKR) surgery and guiding transfusion practice. In this study we aimed to ascertain the optimal timing for Hb determination postoperatively and assess its relationship to serum hematocrit (Hct)., Materials and Methods: This was a retrospective cohort analysis of 61 consecutive patients undergoing preoperative, day 1 and day 2 Hb and Hct concentration determination following TKR surgery. This was a single centre study in the United Kingdom., Results: The mean fall in Hb concentration at day 1 was 2.9 g/dL in comparison to 3.3 g/dL at day 2. This indicated a significant difference of 0.39 g/dL (p=0.023). A total of 5 patients required blood transfusions following day 2 Hb determination. Postoperative Hct values varied in close relation with the Hb concentration with no significant differences demonstrated. Our study reveals a significant change between day 1 and day 2 Hb concentrations following TKR surgery, with no significant differing information provided through Hct determination., Conclusions: Our results support the use of delayed routine testing at day 2 following surgery as it is likely to more accurately reflect ongoing hidden blood loss into the joint cavity and within soft tissue planes.

Disruption in stable establishment of commensal gut microbiota by early weaning is an important factor in susceptibility of young animals to enteric disorders. The artificial sweetener SUCRAM [consisting of neohesperidin dihydrochalcone (NHDC) and saccharin] included in piglets' feed reduces incidence of enteric disease. Pyrosequencing of pig caecal 16S rRNA gene amplicons identified 25 major families encompassing seven bacterial classes with Bacteroidia, Clostridia and Bacilli dominating the microbiota. There were significant shifts in microbial composition in pigs maintained on a diet containing SUCRAM, establishing SUCRAM as a major influence driving bacterial community dynamics. The most notable change was a significant increase of Lactobacillaceae population abundance, almost entirely due to a single phylotype, designated Lactobacillus 4228. The sweetener-induced increase in Lactobacillaceae was observed in two different breeds of pigs signifying a general effect. We isolated Lactobacillus 4228, sequenced its genome and found it to be related to Lactobacillus amylovorus. In vitro analyses of Lactobacillus 4228 growth characteristics showed that presence of NHDC significantly reduces the lag phase of growth and enhances expression of specific sugar transporters, independently of NHDC metabolism. This study suggests that sensing of NHDC by a bacterial plasma membrane receptor underlies sweetener-induced growth of a health promoting gut bacterium., (© 2015 Society for Applied Microbiology and John Wiley & Sons Ltd.)

The interaction of a large number of extracellular proteins with heparan sulfate (HS) regulates their transport and effector functions, but the degree of molecular specificity underlying protein-polysaccharide binding is still debated. The 15 paracrine fibroblast growth factors (FGFs) are one of the paradigms for this interaction. Here, we measure the binding preferences of six FGFs (FGF3, FGF4, FGF6, FGF10, FGF17, FGF20) for a library of modified heparins, representing structures in HS, and model glycosaminoglycans, using differential scanning fluorimetry. This is complemented by the identification of the lysine residues in the primary and secondary binding sites of the FGFs by a selective labelling approach. Pooling these data with previous sets provides good coverage of the FGF phylogenetic tree, deduced from amino acid sequence alignment. This demonstrates that the selectivity of the FGFs for binding structures in sulfated polysaccharides and the pattern of secondary binding sites on the surface of FGFs follow the phylogenetic relationship of the FGFs, and so are likely to be the result of the natural selection pressures that led to the expansion of the FGF family in the course of the evolution of more complex animal body plans., (© 2016 The Authors.)

The aggregation and fibril deposition of amyloid proteins have been implicated in a range of neurodegenerative and vascular diseases, and yet the underlying molecular mechanisms are poorly understood. Here, we use a combination of cell-based assays, biophysical analysis, and atomic force microscopy to investigate the potential involvement of oxidative stress in aortic medial amyloid (AMA) pathogenesis and deposition. We show that medin, the main constituent of AMA, can induce an environment rich in oxidative species, increasing superoxide and reducing bioavailable nitric oxide in human cells. We investigate the role that this oxidative environment may play in altering the aggregation process of medin and identify potential posttranslational modification sites where site-specific modification and interaction can be unambiguously demonstrated. In an oxidizing environment, medin is nitrated at tyrosine and tryptophan residues, with resultant effects on morphology that lead to longer fibrils with increased toxicity. This provides further motivation to investigate the role of oxidative stress in AMA pathogenicity., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

The level of intracellular diadenosine 5', 5'''-P(1),P(4)-tetraphosphate (Ap4A) increases several fold in mammalian cells treated with non-cytotoxic doses of interstrand DNA-crosslinking agents such as mitomycin C. It is also increased in cells lacking DNA repair proteins including XRCC1, PARP1, APTX and FANCG, while >50-fold increases (up to around 25 μM) are achieved in repair mutants exposed to mitomycin C. Part of this induced Ap4A is converted into novel derivatives, identified as mono- and di-ADP-ribosylated Ap4A. Gene knockout experiments suggest that DNA ligase III is primarily responsible for the synthesis of damage-induced Ap4A and that PARP1 and PARP2 can both catalyze its ADP-ribosylation. Degradative proteins such as aprataxin may also contribute to the increase. Using a cell-free replication system, Ap4A was found to cause a marked inhibition of the initiation of DNA replicons, while elongation was unaffected. Maximum inhibition of 70-80% was achieved with 20 μM Ap4A. Ap3A, Ap5A, Gp4G and ADP-ribosylated Ap4A were without effect. It is proposed that Ap4A acts as an important inducible ligand in the DNA damage response to prevent the replication of damaged DNA., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Purpose: To investigate the toxicity and corneal pharmacokinetics of meropenem as a potential antimicrobial for bacterial keratitis., Methods: Corneal epithelial cell and keratocyte toxicity was investigated using methyl thiazolyl tetrazolium (MTT) and LIVE/DEAD assays. The penetration of meropenem through the human cornea was measured using an artificial anterior chamber. In one group of corneas, the epithelial and endothelial layers were removed and in a second group these layers were left intact. We applied 50 μL (10 mg/mL) meropenem to the corneal surface and collected samples in the anterior chamber from 45 minutes up to 24 hours. Meropenem concentrations were estimated with a bioassay and HPLC., Results: Meropenem had significantly higher cellular metabolic activity (MTT assay) at both 5 mg/mL and 2.5 mg/mL compared with moxifloxacin (P = 0.029 and P = 0.018, respectively), with 96% cell viability (LIVE/DEAD assay). The measured values for meropenem concentrations in corneal and aqueous samples were significantly higher using a bioassay than with HPLC (P = 0.004). For both intact and denuded corneas, the concentrations in the anterior chamber increased from 0.48 μg/mL (SD 0.89) and 0.89 μg/mL (SD 0.81) to 6.35 μg/mL (SD 0.81) and 13.48 μg/mL (SD 14.82) using HPLC, and from 0.68 μg/mL (SD 1.50) and 1.31 μg/mL (SD 1.55) to 47.03 μg/mL (SD 5.51) and 43.69 μg/mL (SD 27.22) measured with a bioassay., Conclusions: Meropenem has very low toxicity in vitro. It has good corneal penetration, achieving anterior chamber concentrations above MIC90 for bacteria such as Staphylococcus aureus, Pseudomonas aeruginosa, streptococci, coagulase-negative staphylococci, and the Enterobacteriaceae.

The production of recombinant proteins such as the fibroblast growth factors (FGFs) is the key to establishing their function in cell communication. The production of recombinant FGFs in E. coli is limited, however, due to expression and solubility problems. HaloTag has been used as a fusion protein to introduce a genetically-encoded means for chemical conjugation of probes. We have expressed 11 FGF proteins with an N-terminal HaloTag, followed by a tobacco etch virus (TEV) protease cleavage site to allow release of the FGF protein. These were purified by heparin-affinity chromatography, and in some instances by further ion-exchange chromatography. It was found that HaloTag did not adversely affect the expression of FGF1 and FGF10, both of which expressed well as soluble proteins. The N-terminal HaloTag fusion was found to enhance the expression and yield of FGF2, FGF3 and FGF7. Moreover, whereas FGF6, FGF8, FGF16, FGF17, FGF20 and FGF22 were only expressed as insoluble proteins, their N-terminal HaloTag fusion counterparts (Halo-FGFs) were soluble, and could be successfully purified. However, cleavage of Halo-FGF6, -FGF8 and -FGF22 with TEV resulted in aggregation of the FGF protein. Measurement of phosphorylation of p42/44 mitogen-activated protein kinase and of cell growth demonstrated that the HaloTag fusion proteins were biologically active. Thus, HaloTag provides a means to enhance the expression of soluble recombinant proteins, in addition to providing a chemical genetics route for covalent tagging of proteins.

The 50-amino acid protein medin is the main fibrillar component of human aortic medial amyloid (AMA), the most common form of localised amyloid which affects 97% of Caucasians over the age of 50. Structural models for several amyloid assemblies, including the Alzheimer's amyloid-β peptides, have been defined from solid-state nuclear magnetic resonance (SSNMR) measurements on (13)C- and (15)N-labelled protein fibrils. SSNMR-derived structural information on fibrillar medin is scant, however, because studies to date have been restricted to limited measurements on site-specifically labelled protein prepared by solid-phase synthesis. Here we report a procedure for the expression of a SUMO-medin fusion protein in Escherichia coli and IMAC purification yielding pure, uniformly (13)C,(15)N-labelled medin in quantities required for SSNMR analysis. Thioflavin T fluorescence and dynamic light scattering measurements and transmission electron microscopy analysis confirm that recombinant medin assembles into amyloid-like fibrils over a 48-h period. The first (13)C and (15)N SSNMR spectra obtained for uniformly-labelled fibrils indicate that medin adopts a predominantly β-sheet conformation with some unstructured elements, and provide the basis for further, more detailed structural investigations., (Copyright © 2014. Published by Elsevier Inc.)

Here we describe the identification, purification and characterisation of glycosylated yellow laccase proteins from the basidiomycete fungus Stropharia aeruginosa. Biochemical characterisation of two yellow laccases, Yel1p and Yel3p, show that they are both secreted, monomeric, N-glycosylated proteins of molecular weight around 55kDa with substrate specificities typical of laccases, but lacking the absorption band at 612nm typical of the blue laccase proteins. Low coverage, high throughput 454 transcriptome sequencing in combination with inverse-PCR was used to identify cDNA sequences. One of the cDNA sequences has been assigned to the Yel1p protein on the basis of identity between the translated protein sequence and the peptide data from the purified protein, and the full length gene sequence has been obtained. Biochemical properties, substrate specificities and protein sequence data have been used to discuss the unusual spectroscopic properties of S. aeruginosa proteins in the context of recent theories about the differences between yellow and blue laccases., (Copyright © 2014 Elsevier Inc. All rights reserved.)

A lipase, LipG7, has been purified from the Antarctic filamentous fungus Geomyces sp. P7 which was found to be cold-adapted and able to retain/regain its activity after heat denaturation. The LipG7 exhibits 100% residual activity following 1h incubation at 100°C whilst simultaneously showing kinetic adaptations to cold temperatures. LipG7 was also found to have industrial potential as an enantioselective biocatalyst as it is able to effectively catalyse the enantioselective transesterification of a secondary alcohol. The LipG7 coding sequence has been identified and cloned using 454 pyrosequencing of the transcriptome and inverse PCR. The LipG7 protein has been heterologously expressed in Saccharomyces cerevisiae BJ5465 and shown to exhibit the same characteristics as the native protein., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The culture supernatant fraction of an Enterococcus faecalis gelE mutant of strain OG1RF contained elevated levels of the secreted antigen SalB. Using differential fluorescence gel electrophoresis (DIGE) the salB mutant was shown to possess a unique complement of exoproteins. Differentially abundant exoproteins were identified using matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. Stress-related proteins including DnaK, Dps family protein, SOD, and NADH peroxidase were present in greater quantity in the OG1RF salB mutant culture supernatant. Moreover, several proteins involved in cell wall synthesis and cell division, including d-Ala-d-Lac ligase and EzrA, were present in reduced quantity in OG1RF salB relative to the parent strain. The salB mutant displayed reduced viability and anomalous cell division, and these phenotypes were exacerbated in a gelE salB double mutant. An epistatic relationship between gelE and salB was not identified with respect to increased autolysis and cell morphological changes observed in the salB mutant. SalB was purified as a six-histidine-tagged protein to investigate peptidoglycan hydrolytic activity; however, activity was not evident. High-pressure liquid chromatography (HPLC) analysis of reduced muropeptides from peptidoglycan digested with mutanolysin revealed that the salB mutant and OG1RF were indistinguishable.

Studies of the structural changes invoked in proteins by the binding of the glycosaminoglycan (GAG) polysaccharide portion of proteoglycans are of increasing importance to research in a wide range of fields, from biochemistry and molecular biology to biotechnology and medicine. One important aspect is the degree of stabilisation or destabilisation induced in a protein by the binding of these anionic materials, and this can affect enzyme activity, the stability of complexes, folding and the formation of aggregates, including those in neurodegenerative processes. A simple method, able to determine the effect of interactions with GAG polysaccharides on protein stability is described, based on the propensity of a fluorescent dye-Sypro™ Orange-to present differentiable fluorescence emission spectra following contact with exposed core amino acid residues. The method requires only commonly available and inexpensive equipment and is suitable for a multi-well format, allowing multiple readings to be made simultaneously.

A large body of evidence supports the involvement of heparan sulfate (HS) proteoglycans in physiological processes such as development and diseases including cancer and neurodegenerative disorders. The role of HS emerges from its ability to interact and regulate the activity of a vast number of extracellular proteins including growth factors and extracellular matrix components. A global view on how protein-HS interactions influence the extracellular proteome and, consequently, cell function is currently lacking. Here, we systematically investigate the functional and structural properties that characterize HS-interacting proteins and the network they form. We collected 435 human proteins interacting with HS or the structurally related heparin by integrating literature-derived and affinity proteomics data. We used this data set to identify the topological features that distinguish the heparin/HS-interacting network from the rest of the extracellular proteome and to analyze the enrichment of gene ontology terms, pathways, and domain families in heparin/HS-binding proteins. Our analysis revealed that heparin/HS-binding proteins form a highly interconnected network, which is functionally linked to physiological and pathological processes that are characteristic of higher organisms. Therefore, we then investigated the existence of a correlation between the expansion of domain families characteristic of the heparin/HS interactome and the increase in biological complexity in the metazoan lineage. A strong positive correlation between the expansion of the heparin/HS interactome and biosynthetic machinery and organism complexity emerged. The evolutionary role of HS was reinforced by the presence of a rudimentary HS biosynthetic machinery in a unicellular organism at the root of the metazoan lineage.

The utility of methanesulfonic anhydride for promoting the Friedel-Crafts acylation reaction of aryl and alkyl carboxylic acids is disclosed. This reagent allows the preparation of aryl ketones in good yield with minimal waste containing no metallic or halogenated components, clearly differentiating it from other available methodologies.

The activities of heparan sulfate (HS) and heparin do not correlate simply with sulfation levels or sequence. The alternative hypothesis, that appropriate charge and conformational characteristics for protein binding and activity can be provided by other sequences in heparan sulfate and, possibly, also in unrelated sulfated polysaccharides, is explored. Differential scanning fluorimetry was used to measure the thermostabilisation bestowed by modified heparin polysaccharides (proxies for heparan sulfate) on fibroblast growth factor-1 (FGF-1) and fibroblast growth factor-2 (FGF-2), prototypical heparan sulfate-binding proteins, revealing varied abilities and primary sequence-activity redundancy. The effect of substitution pattern on the heparin/heparan sulfate backbone was explored using principal component analysis of (13)C NMR chemical shift data for homogeneously modified heparin polysaccharides revealing complex conformational effects. No simple relationship emerged between these polysaccharides, with their distinct charge distributions and geometries, and their ability to signal. Other, structurally unrelated sulfated polysaccharides were also able to support signalling. These influenced FGF stabilisation in a similar manner to the HS analogues and provided analogous cell signalling activity. For FGF-1, but not FGF-2, signaling correlated strongly with protein stabilisation and circular dichroism spectroscopy demonstrated that some non-HS polysaccharides invoked comparable secondary structural changes to those induced by heparin. Active conformations can readily be found in several heparin derivatives, as well as among non-HS polysaccharides, which comprise unrelated primary sequences, confirming the hypothesis and implying that the level of unique information contained in HS sequences may be much lower than previously thought.

The interaction between glycosaminoglycans (GAGs) and proteins is important for the regulation of protein transport and activity. Here we present a novel method for the measurement of protein-GAG interactions suitable for high-throughput screening, able to discriminate between the interactions of a protein with GAGs of different structures. Binding of proteins to the GAG heparin, a proxy for sulfated regions of extracellular heparan sulfate, was found to enhance the stability of three test proteins, fibroblast growth factors (FGFs)-1, -2, and -18. Chemically modified heparins and heparin oligosaccharides of different lengths stabilized the three FGFs to different extents, depending on the pattern of sugar binding specificity. The method is based on a differential scanning fluorescence approach. It uses a Sypro Orange dye, which binds to exposed core residues of a denatured protein and results in an increased fluorescence signal. It is convenient, requiring low micromolar amounts of protein and ligand compared to other interaction assays, employing only a real-time polymerase chain reaction (PCR) instrument.

Elevated levels of the calcium-binding protein S100A4 promote metastasis and in carcinoma cells are associated with reduced survival of cancer patients. S100A4 interacts with target proteins that affect a number of activities associated with metastatic cells. However, it is not known how many of these interactions are required for S100A4-promoted metastasis, thus hampering the design of specific inhibitors of S100A4-induced metastasis. Intracellular S100A4 exists as a homodimer through previously identified, well conserved, predominantly hydrophobic key contacts between the subunits. Here it is shown that mutating just one key residue, phenylalanine 72, to alanine is sufficient to reduce the metastasis-promoting activity of S100A4 to 50% that of the wild type protein, and just 2 or 3 specific mutations reduces the metastasis-promoting activity of S100A4 to less than 20% that of the wild type protein. These mutations inhibit the self-association of S100A4 in vivo and reduce markedly the affinity of S100A4 for at least two of its protein targets, a recombinant fragment of non-muscle myosin heavy chain isoform A, and p53. Inhibition of the self-association of S100 proteins might be a novel means of inhibiting their metastasis-promoting activities.

Background/aims: Angiotensin II (AngII) is pivotal in the pathogenesis of progressive kidney disease. We have recently shown that AngII induced an increase in markers of oxidative stress, adaptive responses and upregulated stress-related gene expression in immortalised human proximal tubular (HK-2) cells. However, these observed effects of AngII were not mediated solely via AngII type 1 receptor (ATR1). Both HK-2 cells and primary human renal proximal tubular cells (RPTEC) are useful tools to investigate the renin-angiotensin system (RAS), but data on the local expression of the RAS in these cells remain limited. We therefore characterised RAS expression in RPTEC and HK-2 cells., Methods: The mRNA and protein expression of RAS in RPTEC and HK-2 cells was examined by RT-PCR, Western blotting and immunoprecipitation., Results: In both cell lines, mRNA for angiotensin-converting enzyme (ACE) and mRNA and protein expression for angiotensinogen, renin, ACE2, ATR1 and ATR4 were detected. Candesartan, a specific ATR1 blocker, effectively blocked the expression of 80% of the stress-related genes that were upregulated in HK-2 cells following exposure to AngII., Conclusion: These data support a role for AngII in mediating oxidative stress via other receptor types stimulated by AngII and confirm that it is possible to investigate ATR4 pathways of potential injury in RPTEC., (Copyright © 2010 S. Karger AG, Basel.)

Employing co-catalytic zinc reagents facilitates the iron-catalysed Suzuki cross-coupling of tetraarylborates with both benzyl and 2-heteroaryl halides.

Heparan sulfate proteoglycans are key regulators of complex molecular networks due to the interaction of their sugar chains with a large number of partner proteins, which in humans number more than 200 (Ori, A., Wilkinson, M. C., and Fernig, D. G. (2008) The heparanome and regulation of cell function: structures, functions and challenges. Front. Biosci. 13, 4309-4338). We developed a method to selectively label residues involved in heparin binding that matches the requirements for medium/high throughput applications called the "Protect and Label" strategy. This is based on the protection against chemical modification given by heparin/heparan sulfate to the residues located in the heparin-binding site. Thus, analysis of fibroblast growth factor-2 bound to heparin and incubated with N-hydroxysuccinimide acetate showed that lysines involved in the sugar binding are protected against chemical modification. Moreover following release from heparin, the protected lysine side chains may be specifically labeled with N-hydroxysuccinimide biotin. After protein digestion, the biotinylated peptides were readily isolated and identified by MALDI-Q-TOF mass spectrometry. The analysis of labeled peptides obtained from three well characterized heparin-binding proteins with very different heparin-binding sites, fibroblast growth factor-2, platelet factor-4, and pleiotrophin demonstrates the success of this new approach, which thus provides a rapid and reliable procedure to identify heparin-binding sites.

Iron-based catalysts containing either 1,2-bis(diphenylphosphino)benzene or 1,3-bis(diphenylphosphino)propane give excellent activity and good selectivity in the Negishi coupling of aryl zinc reagents with a range of benzyl halides and phosphates.

The cell-extracellular matrix interface is a crowded space whose structure is dependent on macromolecular assemblies that are dynamic in time, molecular composition and location. Signals travel from one cell to another (or to the same cell) by the regulated assembly/disassembly of molecular complexes. These signals can evoke relatively simple biological responses such cell proliferation and migration, but once integrated, they guide cell fate in complex biological phenomena such as embryonic development and organism homeostasis. Heparan sulfate proteoglycans are ubiquitous components of this space and important actors of these processes in all tissue-organized life forms. A key feature of heparan sulfate is its size, 40 nm to 160 nm, which enables it to integrate self-assembling macromolecular structures over substantial length scales. What is the structure of heparan sulfate? Why do we think heparan sulfate is so important? How do we try to explain its activity? What do we know about its interactions? These questions together with a final look to the future are the "menu" of this review.

Viper venoms contain one of the most potent mixtures of proteases in natural existence and yet the venom gland and proteins in this mixture are refractory to degradation. Here we demonstrate that the sub-10-kDa components of venom from two African viper species (Echis ocellatus and Cerastes cerastes cerastes) are predominantly composed of the tri-peptide pyroglutamate-lysine-tryptophan (pEKW). This tripeptide is encoded by tandemly repeating elements and, in E. ocellatus, on the same transcript as a novel, and highly unusual, poly-histidine-poly-glycine peptide (pHpG) also detected in E. ocellatus venom. The pEKW and pHpG peptides inhibit the proteolytic activity of the haemorrhagic snake venom metalloproteinase (SVMP), EoVMP-2, and the haemorrhagic activity of E. ocellatus venom. These results demonstrate that these vipers express abundant transcripts encoding tandemly repeated protease inhibitor cassettes and accumulate significant quantities of peptide inhibitors in venoms to provide a basis for attenuating the proteolytic activity of SVMPs.

Salivary gland proteins of the human malaria vector, Anopheles dirus B were determined and analyzed. The amount of salivary gland proteins in mosquitoes aged between 3--10 days was approximately 1.08 +/- 0.04 microg/female and 0.1 +/- 0.05 microg/male. The salivary glands of both sexes displayed the same morphological organization as that of other anopheline mosquitoes. In females, apyrase accumulated in the distal regions, whereas alpha-glucosidase was found in the proximal region of the lateral lobes. This differential distribution of the analyzed enzymes reflects specialization of different regions for sugar and blood feeding. SDS-PAGE analysis revealed that at least seven major proteins were found in the female salivary glands, of which each morphological region contained different major proteins. Similar electrophoretic protein profiles were detected comparing unfed and blood-fed mosquitoes, suggesting that there is no specific protein induced by blood. Two-dimensional polyacrylamide gel analysis showed the most abundant salivary gland protein, with a molecular mass of approximately 35 kilodaltons and an isoelectric point of approximately 4.0. These results provide basic information that would lead to further study on the role of salivary proteins of An. dirus B in disease transmission and hematophagy.

Before transmission, malaria parasites reside in the salivary glands of their female mosquito hosts. Saliva proteins assist in blood feeding and also may influence the ability of mosquitoes to transmit malaria. We attempted to identify and isolate cDNAs encoding proteins expressed at a high level in the salivary glands of a malaria vector, Anopheles dirus B Peyton and Harrison (= An. cracens) (Diptera: Culicidae). A major protein with an estimated molecular mass of 35 kDa and an isoelectric point (pI) of approximately 4 was detected on a two-dimensional (2D) gel. Internal peptide sequences of the protein spot showed high similarity to sequences present in the conserved C-terminal domain of glycine- and glutamate (GE)-rich proteins. A full-length cDNA encoding this protein was isolated from a salivary gland cDNA library of female An. dirus B. The cDNA encoded a 256-residue protein with a calculated molecular mass of 25.4 kDa and a pI of 3.9. BLAST analysis confirmed that it is a member of the GE-rich family. Compositional and sequence analysis of this and other family members revealed a highly acidic N-terminal region of variable length and low sequence conservation and a well conserved C-terminal domain containing 10 identical residues across the 13 known members of the gene family in mosquitoes. The An. dirus B GE-rich transcript was detected by reverse transcription-polymerase chain reaction (PCR) only in the female salivary glands, indicating that this protein is female saliva-specific. The GE-rich proteins may function as a salivary lubricant to facilitate blood feeding.

In this study, we isolated a novel prothrombin activator from the venom of Bothrops cotiara, a Brazilian lance-headed pit viper (Cotiara, Jararaca preta, Biocotiara), which we have designated "cotiaractivase" (prefix: cotiar- from B. cotiara; suffix: -activase, from prothrombin activating activity). Cotiaractivase was purified using a phenyl-Superose hydrophobic interaction column followed by a Mono-Q anion exchange column. It is a single-chain polypeptide with a molecular weight of 22,931 Da as measured by mass spectroscopy. Cotiaractivase generated active alpha-thrombin from purified human prothrombin in a Ca2+-dependent manner as assessed by S2238 chromogenic substrate assay and SDS-PAGE. Cotiaractivase cleaved prothrombin at positions Arg271-Thr272 and Arg320-Ile321, which are also cleaved by factor Xa. However, the rate of thrombin generation by cotiaractivase was approximately 60-fold less than factor Xa alone and 17 x 10(6)-fold less than the prothrombinase complex. The enzymatic activity of cotiaractivase was inhibited by the chelating agent EDTA, whereas the serine protease inhibitor PMSF had no effect on its activity, suggesting that it is a metalloproteinase. Interestingly, S2238 inhibited cotiaractivase activity non-competitively, suggesting that this toxin contains an exosite that allows it to bind prothrombin independently of its active site. Tandem mass spectrometry and N-terminal sequencing of purified cotiaractivase identified peptides that were identical to regions of the cysteine-rich and disintegrin-like domains of known snake venom metalloproteinases. Cotiaractivase is a unique low molecular weight snake venom prothrombin activator that likely belongs to the metalloproteinase family of proteins.

An N-acetyl-D-galactosamine (GalNAc)-specific lectin was purified from the edible mushroom, Schizophyllum commune, using affinity chromatography on a porcine stomach mucin (PSM)-Sepharose 4B column. Under reducing and non-reducing conditions, SDS-polyacrylamide gel electrophoresis gave a major band of 31.5 kDa. The Schizophyllum commune lectin (SCL) showed high affinity toward rat erythrocytes and the sugar inhibition assay exhibited its sugar specificity highly toward lactose and N-acetyl-D-galactosamine. It was stable at 55 degrees C for 30 min and at pH 3-10 for 18-h test. The lectin was shown to be a glycoprotein with cytotoxic activity against human epidermoid carcinoma cells. The N-terminus of SCL was blocked but amino acid sequences of internal tryptic peptides showed moderately sequence similarities with some other fungal and plant lectins. Crystals of SCL were obtained by the sitting drop vapour-diffusion method using polyethylene glycol 8000 as the precipitant, and gave an X-ray diffraction pattern to approximately 3.8 angstroms resolution.

Many toxins from viperid venoms have been characterised as powerful activators of platelets. Here, the venom from the East African Lowland viper, Proatheris superciliaris, was investigated for its effect on platelets and the coagulation system. Whole P. superciliaris venom stimulated platelet shape change and aggregation; however, the stimulation of platelet activation was unaffected by the structurally distinct Src family kinase inhibitors PP1 and PD0173952, suggesting that platelet activation was mediated by a G protein-coupled receptor. A platelet reactive 34-kDa protein was isolated from P. superciliaris venom which we have designated proatherocytin. This protein induced Src kinase-independent aggregation of both human and mouse platelets that was inhibited by the serine protease inhibitor AEBSF. Proatherocytin did not clot bovine or human fibrinogen, degrade fibrinogen or hydrolyse the serine protease substrate benzoyl-FVR-pNA. It activated human PAR1 on stably transfected rat kidney epithelial cells, whereas no activation of the trypsin receptor PAR2 was shown. Surprisingly, Edman degradation of proatherocytin revealed sequence identity with existing disintegrin-like domains of snake venom metalloproteinases. These results suggest that proatherocytin is a highly selective PAR1 agonist that also causes mouse platelet aggregation, probably through cleavage of PAR4.